Diverse sets of sensory afferents that transmit temperature, touch and painful stimuli innervate the epidermal layer of the skin. Although stimulus detection was thought to be afferent specific, production of growth factors (NGF), neuromodulators (e.g., ATP, ACh, CGRP) and activation of ion channels (e.g., TRP, Na+) by epidermal keratinocytes suggests the epidermis also impacts sensory signaling. To examine nerve-epithelial communication we first developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to peripheral neurons using a peripherin promoter driving Cre recombinase. Light stimulation of the skin of peripherin-ChR2 mice elicits a robust nocifensive behavioral response and electrophysiological assays using a skin-nerve-ganglia and spinal cord ex vivo preparation showed preferential activation of C-fiber nociceptors. Thus, blue-laser light penetrates the epidermis and sensory neuron specific gene promoters drive in vivo expression of ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to thoe obtained using direct mechanical or thermal stimulation of the skin. In this R21 application we therefore propose to test the hypothesis that the difference between light-stimulation and direct mechanical or thermal stimulation of skin is due to factors released by the skin in response to mechanical or thermal manipulation. To test this hypothesis we isolated optogenetic mouse strains in which ChR2 is targeted to epidermal keratinocytes using the K14 keratin gene promoter. Preliminary analysis of these mice using an ex vivo skin-nerve-ganglia-spinal cord preparation show that light stimulation of keratinocytes evokes changes in behavioral and electrophysiologic response properties. We also found that different subtypes of cutaneous afferents are activated and that combined stimulation of the skin using light and mechanical or thermal stimuli evokes greater activation of individual fibers. In Aim 1 we will further characterie K14-ChR2 mice at anatomical, behavioral and biochemical levels. Aim 2 experiments will determine how light activation of keratinocytes affects response properties of functionally defined subsets of cutaneous sensory neurons and determine how this activation compares to mechanical and/or thermal stimulation of the skin. We will use the data generated in this R21 pilot study to design future studies that will investigate neuromodulator release mechanisms and determine if neuromodulators released from keratinocytes activate specific sensory afferent subtypes.